| 应用分子伴侣共表达系统表达pfu基因及酶活性测定 |
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| 引用本文： | 张海军,杨君,刘晓光,胡向阳.应用分子伴侣共表达系统表达pfu基因及酶活性测定[J].云南植物研究,2009,31(6):499-503. |
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| 作者姓名： | 张海军 杨君 刘晓光 胡向阳 |
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| 作者单位： | 1. 江苏大学生命科学研究院,镇江,江苏,212013
2. 中国科学院昆明植物研究所,云南,昆明,650204;中国科学院青藏高原研究所昆明部,云南,昆明,650204 |
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| 基金项目： | 国家自然科学基金面上项目，中国科学院百人计划项目 |
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| 摘 要： | 将通过In-fution方法构建的pET32a-pfu质粒与可以促进可溶性表达的HG-PGR07质粒一起转入大肠杆菌B121（DE3）共表达，以pET32a-pfu单独在B121（DE3）中表达作为对照。用热变性和（NH4）2SO4沉淀去除部分杂蛋白，再经Ni—NAT亲和层析柱纯化分离尼血蛋白，SDS-PAGE检测结果表明目的蛋白大小约为90kD，与预计的分子量大小一致。最后对其酶活性测定结果表明分子伴侣能够促进pfu基因表达，并能够提高酶活性。
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| 关 键 词： | Pfu 分子伴侣 基因克隆 载体构建 原核表达 蛋白纯化 酶活测定 |
Expression and Enzyme Activity Assay of pfu with Molecular Chaperone |
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| ZHANG Hai-Jun,YANG Jun,LIU Xiao-Guang,HU Xiang-Yang.Expression and Enzyme Activity Assay of pfu with Molecular Chaperone[J].Acta Botanica Yunnanica,2009,31(6):499-503. |
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| Authors: | ZHANG Hai-Jun YANG Jun LIU Xiao-Guang HU Xiang-Yang |
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| Institution: | ZHANG Hai-Jun , YANG Jun , LIU Xiao-Guang , HU Xiang-Yang (1 Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China; 2 Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China; 3 Institute of Tibetan Plateau Research at Kunming , Chinese Academy of Sciences, Kunming 650204, China) |
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| Abstract: | Co-expressing the plasmid pET32a-pfu which was constrcted using In-fution technique with chaperone plasmid HG-PGR07 at the same time in E.coli.BL21(DE3).The expression system,which expressed pfu gene alone in E.coli.BL21(DE3),was used as control.Most of unnecessary proteins were exenterated by heat treatment and(NH_4)_2SO_4 precipi-tation.The purified fusion protein was obtained by the Ni chelating resin affinity chromatography.The SDS-PAGE analysis showed that the molecular mass of the purified fusion protein was about 90kD.conforming to the expected molecular mass of Pfu protein.The results of enzyme activity assay of pfu demonstrated that molecular chaperone was able to activate pfu gene expression and its enzyme activity. |
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| Keywords: | Pfu Pfu Molecular chaperone Clonging Construction of a Expression Vector Prokaryotic expression Protein purification Enzyme activity assay |
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